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SON and SRRM2 are essential for nuclear speckle formation.

eLife (2020-10-24)
İbrahim Avşar Ilik, Michal Malszycki, Anna Katharina Lübke, Claudia Schade, David Meierhofer, Tuğçe Aktaş
ABSTRACT

Nuclear speckles (NS) are among the most prominent biomolecular condensates. Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified. The monoclonal antibody SC35, raised against a spliceosomal extract, is frequently used to mark NS. Unexpectedly, we found that this antibody was mischaracterized and the main target of SC35 mAb is SRRM2, a spliceosome-associated protein that sharply localizes to NS. Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS, while co-depletion of SON and SRRM2 or depletion of SON in a cell-line where intrinsically disordered regions (IDRs) of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2-((Di-tert-butylphosphinomethyl)-6-diethylaminomethyl)pyridine
Sigma-Aldrich
Anti-SON antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Monoclonal Anti-Splicing Factor SC-35 antibody produced in mouse, clone SC-35, ascites fluid
Sigma-Aldrich
MISSION® esiRNA, targeting human SRRM2
Sigma-Aldrich
Anti-RBM25 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody
Sigma-Aldrich
Poly-L-lysine hydrobromide, mol wt 30,000-70,000, lyophilized powder, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
MISSION® esiRNA, targeting human SON
Sigma-Aldrich
MISSION® esiRNA, targeting human RBM25
Sigma-Aldrich
Anti-SC-35 Antibody, clone 1SC-4F11, ascites fluid, clone 1SC-4F11, from mouse
Sigma-Aldrich
MISSION® esiRNA, targeting human SRRM1